New Models to Assay Gene Silencing Therapies

描述(由申请人提供)：在过去的几年里，人们越来越多地探索基因沉默或敲除疗法治疗神经退行性疾病的可能性。这项技术已经取得进展，已经启动了针对家族性肌萎缩侧索硬化症的1期人类治疗试验。目前正在研究的击倒疗法包括病毒载体递送shRNAi或microRNA模拟物，递送裸露的RNAi和RNAi与各种试剂复合以促进摄取，以及递送修饰的反义DNA寡核苷酸。这些途径的作用机制包括调节mRNA的翻译、调节Pre-mRNA的剪接、以及降解mRNA和Pre-mRNA。这些不同的方法已经在临床前动物模型中进行了不同程度的测试，取得了不同程度的疗效。到目前为止进行的这些研究的一个明显局限性是，通常不可能实时监测击倒的效果。为了克服这一限制，我们提出了两个目标，旨在建立实时跟踪基因敲除有效性的能力，并在两种神经退行性疾病的小鼠模型中提供概念研究的证据，这两种疾病是基因沉默努力的潜在目标。利用相关研究人员的专业知识，我们计划将重点放在亨廷顿S病和额颞部痴呆的模型上。这些疾病是基因沉默疗法的主要候选者，之前在小鼠身上模拟这些疾病的工作已经产生了概括每种疾病的方面的模型。在这里描述的方法中，我们寻求生成模型，在该模型中，我们将产生可分析和可观察的行为表型，同时能够实时监测基因沉默试剂的有效性。在目标1中，我们将产生表达突变形式的人tau的小鼠，该突变形式的人tau融合到荧光素酶的框内。在目标2中，我们将类似地产生表达突变的亨廷顿蛋白N端片段的小鼠，该片段与荧光素酶的框内融合。在这两个构建中，我们将使用一种技术，促进多聚蛋白的翻译后处理，以释放荧光素酶，以便可以独立于突变tau或Huntingtin的病理性积累来检测它。我们建议使用新的体内成像技术来检测和测量由表达的荧光素酶催化的生物发光。在我们建议建立的tau模型中，我们预计动物会出现可测量的记忆缺陷，并伴有神经病理异常，包括神经元丢失和神经原纤维缠绕。 病理学。在亨廷顿S模型中，我们同样希望诱导出可分析的表型，包括运动功能缺陷、纹状体部分基因转录减少、活动不足和过早死亡。因此，人们最终可以拥有具有双重读出能力的模型，在该模型中，可以通过监测荧光素酶的活性水平来实时监测表达的减少，同时还可以检测与疾病相关的表型。 公共卫生相关性：本申请中提出的研究旨在产生和表征新的动物模型，用于对难治性人类神经退行性疾病的基因沉默方法进行临床前测试。我们提出的创新模型将允许在体内实时评估基因沉默或基因敲除疗法的有效性，使研究人员能够进入第一阶段临床试验，获得最优化和最特异的治疗方法。 免责声明：请注意，以下批评是由评审员在研究小组会议之前准备的，基本上是以未经编辑的形式提供的。虽然审查员有机会根据小组的讨论更新或修订其书面评价，但不能保证在会议讨论之后更新了个别批评意见。因此，这些评论可能不能完全反映评审员在小组讨论结束时的最终意见或小组的最终多数意见。因此，讨论纪要和总结是审查员在会议上实际上认为至关重要的最后结论。

DESCRIPTION (provided by applicant): Over the past few years, there has been increasing exploration of the potential for gene silencing or knockdown therapies in the treatment of neurodegenerative disorders. The technology has progressed to a point in which a phase 1 human therapeutic trial for familial amyotrophic lateral sclerosis has been initiated. Current knock-down therapies under investigation include viral vector delivery of shRNAi or microRNA mimics, delivery of naked RNAi and RNAi complexed with various reagents to facilitate uptake, and delivery of modified antisense DNA oligonucleotides. The mechanisms of action for these approaches include modulation of mRNA translation, modulation of pre-mRNA splicing, and degradation of mRNA and pre-mRNA. These various approaches have been tested in pre-clinical animal models to varying extents with varying levels of efficacy. A glaring limitation of these studies that have been conducted thus far is that it has generally been impossible to monitor the efficacy of knock-down in real time. To overcome this limitation, we propose two Aims that are designed to build capability to track the efficacy of knock-down in real time and provide proof of concept studies in mouse models of two neurodegenerative diseases that are potential targets for gene silencing efforts. Taking advantage of the expertise of the investigators involved, we plan to focus on models for Huntington s disease and fronto-temporal dementia. These disorders are prime candidates for gene-silencing therapeutics and previous work in modeling these disorders in mice has produced models that recapitulate aspects of each disorder. In the approach described here, we seek to generate models in which we will produce assayable and observable behavioral phenotypes while simultaneously being able to monitor the efficacy of gene silencing reagents in real-time. In Aim 1, we will generate mice that express mutant forms of human tau fused in-frame to luciferase. In Aim 2, we will similarly generate mice that express mutant Nterminal fragments of huntingtin fused in-frame to luciferase. In both constructs we will employ a technique that facilitates post-translational processing of the poly-protein to liberate the luciferase so it can be assayed independently of pathologic accumulations of mutant tau or huntingtin. We propose to use new in vivo imaging techniques to detect and measure bioluminescence catalyzed by the expressed luciferase. In the tau model we propose to generate, we expect the animals to develop measurable memory deficits with neuropathological abnormalities that include neuronal loss and neurofibrillary tangle pathology. In the Huntington s model, we similarly expect to induce assayable phenotypes, which include motor function deficits, reduction in the transcription of a subset of genes in striatum, hypoactivity, and premature death. Thus, one could ultimately have models with dual readout capability in which reductions in expression could be monitored in real-time by monitoring luciferase activity levels while simultaneously having disease-relevant phenotypes to assay. PUBLIC HEALTH RELEVANCE: The studies proposed in this application are designed to produce and characterize new animal models for pre-clinical testing of gene silencing approaches for intractable human neurodegenerative disease. The innovative models we propose will allow real-time, in vivo, assessments of the efficacy of gene silencing or gene knockdown therapies, allowing investigators to move to Phase I clinical trials with the most optimized and specific therapeutic available. Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of th individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.